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Evolution of the Muscarinic Acetylcholine Receptors in Vertebrates and Dan Larhammar ء,Christina A

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Evolution of the Muscarinic Acetylcholine Receptors in Vertebrates and Dan Larhammar ء,Christina A New Research Development Evolution of the Muscarinic Acetylcholine Receptors in Vertebrates and Dan Larhammar ء,Christina A. Bergqvist ء,Julia E. Pedersen https://doi.org/10.1523/ENEURO.0340-18.2018 Department of Neuroscience, Unit of Pharmacology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 24, Sweden Abstract The family of muscarinic acetylcholine receptors (mAChRs) consists of five members in mammals, encoded by the CHRM1-5 genes. The mAChRs are G-protein-coupled receptors, which can be divided into the following two subfamilies: M2 and M4 receptors coupling to Gi/o; and M1, M3, and M5 receptors coupling to Gq/11. However, despite the fundamental roles played by these receptors, their evolution in vertebrates has not yet been fully described. We have combined sequence-based phylogenetic analyses with comparisons of exon–intron organi- zation and conserved synteny in order to deduce the evolution of the mAChR receptors. Our analyses verify the existence of two ancestral genes prior to the two vertebrate tetraploidizations (1R and 2R). After these events, one gene had duplicated, resulting in CHRM2 and CHRM4; and the other had triplicated, forming the CHRM1, CHRM3, and CHRM5 subfamily. All five genes are still present in all vertebrate groups investigated except the CHRM1 gene, which has not been identified in some of the teleosts or in chicken or any other birds. Interestingly, the third tetraploidization (3R) that took place in the teleost predecessor resulted in duplicates of all five mAChR genes of which all 10 are present in zebrafish. One of the copies of the CHRM2 and CHRM3 genes and both CHRM4 copies have gained introns in teleosts. Not a single separate (nontetraploidization) duplicate has been identified in any vertebrate species. These results clarify the evolution of the vertebrate mAChR family and reveal a doubled repertoire in zebrafish, inviting studies of gene neofunctionalization and subfunctionalization. Key words: acetylcholine; G-protein-coupled receptor; gene duplication; muscarinic; tetraploidization; zebrafish Significance Statement Despite their pivotal physiologic role, the evolution of the muscarinic acetylcholine receptors (mAChRs) has not yet been resolved. By investigating the genomes of a broad selection of vertebrate species and combining three different types of data, namely sequence-based phylogeny, conserved synteny, and intron organization, we have deduced the evolution of the mAChR genes in relation to the major vertebrate tetraploidizations (1R, 2R, and 3R). Our analyses show that all vertebrate mAChR gene duplications resulted from the tetraploidizations. Interestingly, following 3R, zebrafish doubled its gene number, resulting in the 10 mAChR genes present. By knowing how and when the mAChR genes arose, studies of receptor subtype specialization and possible neofunctionalization or subfunctionalization can follow. Introduction memory. They are also present in the peripheral nervous The muscarinic acetylcholine receptors (mAChRs) are system and smooth muscle tissue. The mAChR family G-protein-coupled receptors (GPCRs) involved in a vari- consists of five different receptor subtypes named M1– ety of CNS processes such as cognition, learning, and M5, which are encoded by the CHRM1-5 genes. The Received August 27, 2018; accepted October 17, 2018; First published Author contributions: J.E.P., C.A.B., and D.L. designed the research. J.E.P. October 24, 2018. and C.A.B. performed research; J.E.P., C.A.B., and D.L. analyzed data; J.E.P. The authors declare no competing financial interests. and D.L. wrote the paper. September/October 2018, 5(5) e0340-18.2018 1–12 New Research 2 of 12 structures of the muscarinic receptors follow the typical is important to deduce evolutionary relationships to dis- GPCR structure with the extracellular N terminus followed tinguish orthologs (species homologs), paralogs (gene by seven transmembrane (TM) domains (TM domains duplicates), and ohnologs (gene duplicates resulting spe- 1–7), which are separated by three intracellular loops (ILs; cifically from tetraploidization events), especially when 1–3), three extracellular loops (ELs; 1–3), and finally the studying species that belong to evolutionarily distant intracellular C terminus. The orthosteric binding site for groups, for instance the commonly used experimental acetylcholine consists of a hydrophobic pocket formed by animals mouse/rat, chicken, and zebrafish. Furthermore, the side chains of TM domains 3–7. The crystal structures the time points of the gene duplication events are impor- of the M2 and M3 receptors have been reported (Haga tant for studies of evolutionary change between orthologs et al., 2012; Kruse et al., 2012), showing that the binding and paralogs as well as ohnologs. It is now well estab- pocket contains identical amino acid residues in the M2 lished that the vertebrate predecessor underwent two and M3 receptors (Haga et al., 2012; Kruse et al., 2012; rounds of whole-genome duplication (i.e., tetraploidiza- Tautermann et al., 2013). In the study by Haga et al. tions) before the radiation of jawed vertebrates (Nakatani (2012), 14 amino acid residues were found to form the et al., 2007; Putnam et al., 2008). These two events are antagonist binding sites and, following modeling of ace- usually referred to as 1R and 2R. In addition, the ancestor tylcholine into the antagonist-binding pocket, 6 of these of the teleosts went through a third tetraploidization (3R) residues were suggested to bind acetylcholine. These six after the divergence from the most basal lineages of proposed acetylcholine-binding residues have also been ray-finned fishes (Jaillon et al., 2004). reported to be conserved in Drosophila melanogaster As the availability of high-quality genome assemblies is (Collin et al., 2013). In the EL regions, the amino acid continuously increasing, it is now possible to perform a more residues are less conserved, hence these have been tar- extensive analysis of the evolution of the mAChR family. gets for the design of drugs working as allosteric modu- We have used an approach that combines amino acid lators (Christopoulos, 2002; Kruse et al., 2013, 2014). The sequence-based phylogeny and analyses of chromosomal M1, M3, and M5 receptors form one subfamily, coupling locations for comparison of synteny and duplicated chromo- to Gq/11, and the M2 and M4 receptors form one subfam- some regions. We report here that the 1R and 2R genome- ily, coupling to Gi/o. Hence, acetylcholine may give rise to doubling events duplicated the two ancestral mAChR genes different responses depending on which receptor subtype to the five genes that are present today in all tetrapods is present to initiate the signal transduction. investigated except birds, where CHRM1 has not been The mAChRs are widely expressed in the nervous sys- found. Furthermore, the teleost 3R event doubled the rep- tem, the cardiovascular system, and the gastrointestinal ertoire once more, resulting in the 10 genes present today in tract, as well as elsewhere. In the peripheral nervous zebrafish, albeit some teleosts seem to lack both copies of system, the mAChRs play a major role in the parasympa- CHRM1. This long-lived multiplicity invites further studies of thetic system stimulating smooth muscle contraction and the roles of each of the subtypes. glandular secretion as well as slowing the heart rate (Eg- len, 2005). In the CNS of primates and rodents, the M1, Materials and Methods M2, and M4 receptors are the most highly expressed mAChRs in the brain, but M3 and M5 are also present Species included in analysis and amino acid (Thiele, 2013; Lebois et al., 2018). Regarding mechanisms sequence retrieval behind gene expression and similarities or dissimilarities Species sequences included in the analysis of the among vertebrate species, little is known (Lebois et al., mAChR family were the human (Homo sapiens; Hsa), 2018). Each gene may have multiple promoters as has mouse (Mus musculus; Mmu), opossum (Monodelphis been demonstrated for CHRM2 (Krejci et al., 2004), and domestica; Mdo), chicken (Gallus gallus; Gga), anole lizard while some promoters are conserved across mammals, (Anolis carolinensis; Aca), frog (Xenopus tropicalis; Xtr), others differ and presumably contribute to anatomic or coelacanth (Latimeria chalumnae; Lch), spotted gar (Lep- temporal differences in expression between species. isosteus oculatus; Loc), Japanese eel (Anguilla japonica; Although the muscarinic receptors have prominent Aja), European eel (Anguilla anguilla; Aan), zebrafish roles in various nervous system functions, the evolution of (Danio rerio; Dre), stickleback (Gasterosteus aculeatus; the mAChR gene family has not yet been fully resolved. It Gac), medaka (Oryzias latipes; Ola), tunicates (Ciona in- testinalis; Cin and Ciona savigny; Csa), and nematode (Caenorhabditis elegans; Cel). The amino acid sequences This project was supported by grants from the Carl Trygger Foundation and from the species listed were retrieved from the Ensembl the FACIAS Foundation. *J.E.P. and C.A.B contributed equally. genome browser (release 87; Zerbino et al., 2018; En- We thank Jan-Erik Borg for preliminary analyses at the initial stage of this sembl Genome Browser, RRID:SCR_013367) or NCBI project. (NCBI; RRID:SCR_006472) databases. If sequences were Correspondence should be addressed to Dan Larhammar, Department of Neuro- not found in either of the databases, the sequence of a science, Unit of Pharmacology, Science for Life Laboratory, Box 593, Uppsala
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